Immunoprecipitation Protocol Utilizing Magnetic Separation (For Analysis By Western Immunoblotting)

A. Solutions and Reagents

NOTE: Prepare solutions with Milli-Q or equivalently purified water.

1. 20X Phosphate Buffered Saline (PBS) #9808: To prepare 1 L of 1X PBS, add 50 mL 20X PBS to 950 mL dH2O, mix.
2. 10X Cell Lysis Buffer #9803: To prepare 10 mL of 1X cell lysis buffer, add 1 mL 10X cell lysis buffer to 9 mL dH2O, mix.

   NOTE: Add 1 mM PMSF #8553 immediately prior to use.

3. Protein A or G Magnetic Beads: Use Protein A #73778 for rabbit pull down and Protein G #70024 for mouse IgG pull down.
4. 3X SDS Sample Buffer: Use Blue Loading Buffer Pack #7722 or Red Loading Buffer Pack #7723. Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
5. Magnetic Separation Rack: Use #7017 or #14654.

B. Preparing Cell Lysates

1. Aspirate media. Treat cells by adding fresh media containing a regulator for the desired time.
2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
3. Remove PBS and add 0.5 mL ice-cold 1X cell lysis buffer to each plate (10 cm), then incubate the plates on ice for 5 min.
4. Scrape cells off the plates and transfer to microcentrifuge tubes. Keep on ice.
5. Sonicate samples on ice three times for 5 sec each.
6. Microcentrifuge for 10 min at 4°C, 14,000 x g, then transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Highly Recommended)

A cell lysate pre-clearing step is highly recommended to reduce nonspecific protein binding to the Protein A or G magnetic beads. Pre-clear enough lysate for test samples and isotype controls.

1. Briefly vortex the stock tube to resuspend the magnetic beads.

   IMPORTANT: Pre-wash the magnetic beads just prior to use.

2. Transfer 20 µL of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 sec.

   Carefully remove the buffer once the solution is clear. Add 500 µL of 1X cell lysis buffer to the magnetic bead pellet, then briefly vortex to wash the beads. Place the tube back in a magnetic separation rack. Remove the buffer once the solution is clear. Repeat washing step once more.

3. Add 200 µL cell lysate to 20 µL of pre-washed magnetic beads.

   IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration range of 250 µg/mL - 1.0 mg/mL is recommended.

4. Incubate with rotation for 20 min at room temperature.
5. Separate the magnetic beads from the lysate using a magnetic separation rack. Transfer the pre-cleared lysate to a clean tube. Discard the magnetic bead pellet.

Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Isotype controls should be concentration-matched and run alongside the primary antibody samples.

1. Add primary antibody at the appropriate dilution, as recommended on the product webpage or datasheet, to 200 µL cell lysate. Incubate with rotation overnight at 4°C to form the immunocomplex.

   IMPORTANT: For proteins prone to degradation, such as high molecular weight proteins, a 2-4 hr incubation at 4°C may be optimal.

2. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2).
3. Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet.
4. Incubate with rotation for 20 min at room temperature.
5. Pellet magnetic beads by placing the tubes in a magnetic separation rack #7017 or #14654. Wait 1-2 min for the solution to clear. Wash pellet five times with 500 µL of 1X cell lysis buffer. Keep on ice between washes.
6. Resuspend the pellet in 20-40 µL 3X SDS sample buffer. Briefly vortex to mix, then microcentrifuge for 30 sec.
7. Heat the sample to 95-100°C for 5 min.
8. Pellet magnetic beads by placing the tubes in a magnetic separation rack #7017 or #14654. Wait 1-2 min for the solution to clear. Transfer the supernatant to a new tube. The supernatant is the sample.
9. Load the sample (15-35 µL) on SDS-PAGE gel.
10. Analyze the sample by western blotting. Please refer to the Western Blotting Protocol.

NOTE: To minimize masking of target bands produced by denatured heavy chains or interference produced by denatured light chains, the following secondaries are suggested:

For proteins with molecular weights at approximately 50 kDa:

Mouse Anti-rabbit IgG (Light-Chain Specific) (D4W3E) Monoclonal Antibody #45262

Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) Monoclonal Antibody (HRP Conjugate) #93702

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) Monoclonal Antibody #3678

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) Monoclonal Antibody (HRP Conjugate) #5127

For proteins with molecular weights at approximately 25 kDa:

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) Monoclonal Antibody #3678

Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) Monoclonal Antibody (HRP Conjugate) #5127